Xylitol is a polyalcohol or sugar alcohol (alditol) of formula (CHOH)3(CH2OH)2, that has applications in hygiene and nutraceutical formulations and products.
Xylitol is used as a diabetic sweetener which is roughly as sweet as sucrose with 33% fewer calories. Unlike other natural or synthetic sweeteners, xylitol is actively beneficial for dental health by reducing caries to a third in regular use and helpful to remineralization.
Xylitol is naturally found in low concentrations in the fibers of many fruits and vegetables, and can be extracted from various berries, oats, and mushrooms, as well as fibrous material such as corn husks and sugar cane bagasse, and birch.
However, industrial production starts from xylan (a hemicellulose) extracted from hardwoods or corncobs, which is hydrolyzed into xylose and catalytically hydrogenated into xylitol.
Purification of xylose and also xylitol presents therefore a significant problem. A number of processes of this type are known. U.S. Pat. Nos. 4,075,406 and 4,008,285 can be mentioned as examples.
The reduction of D-xylose into xylitol can also be achieved in a microbiological process using either yeast strains isolated from nature (wild type strains) or genetically engineered strains.
However, obtaining the substrate, D-xylose, in a form suitable for yeast fermentation is a problem because inexpensive xylose sources such as sulphite liquor from pulp and paper processes contain impurities which inhibit yeast growth.
An attractive alternative method for the manufacture of xylitol is obtaining it by fermentation of a cheap and readily available substrate, such as D-glucose.
In the state of the art, there are some recombinant microorganisms described able to produce xylitol in certain amounts during a one-step fermentation of any common carbon sources other than D-xylose and D-xylulose.
These recombinant microorganisms, especially osmophilic yeasts, are for example Zygosaccharomyces rouxii, Candida polymorpha, and Torulopsis candida, initially known as producers of significant amounts of a xylitol closely related pentitol, which is D-arabitol, from D-glucose (Lewis D. H. & Smith D. C., 1967, New Phytol. 66:143-184).
Thus, the international patent application WO 94/10325 provides methods for constructing such recombinant hosts being capable of producing xylitol when grown on carbon sources other than D-xylulose or D-xylose, and other than polymers or oligomers or mixtures thereof.
In the current patent application, this goal is achieved through modification of the metabolism of the desired microorganism, preferably a naturally occurring yeast microorganism, by introducing and expressing desired heterologous genes.
This goal is also achieved by further modification of the metabolism of such desired microorganism, so as to overexpress and/or inactivate the activity or expression of certain genes homologous to such microorganism in its native state.
The method provided in this patent application for the production of xylitol utilized an altered D-arabitol biosynthesis pathway, and such pathway being notably altered by extending the preexisting D-arabitol pathway by the introduction and overexpression of the genes coding for D-xylulose-forming D-arabitol dehydrogenase (EC 1.1.1.11) and xylitol dehydrogenase (EC 1.1.1.9) into an D-arabitol-producing microorganism.
However, the yield of xylitol in the trials described in WO 94/10325 was only approximately 7.7 g/l after 48 hours of cultivation in a medium with yeast extract.
To try to optimize this first result, it was further proposed in WO 94/10325 to inactivate, using mutagenesis or gene disruption, the genes coding for transketolase (EC 2.2.1.1) and/or the gene coding for D-xylulokinase (EC 2.7.1.17), and also to overexpress the genes coding for the enzymes of the oxidative branch of the pentose-phosphate pathway, and specifically D-glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and/or 6-phospho-D-gluconate dehydrogenase (EC 1.1.1.44) and/or D-ribulose-5-phosphate epimerase gene (EC 5.1.3.1) in such microorganisms.
But, whatever the genetic combination employed, the xylitol titer was never more than 9 g/l.
There is therefore still an unsatisfied need for a better genetic manipulation of xylitol producing strains in order to optimize its production, and thus make it commercially profitable.